Blood cells originate from a self-renewing population of hemopoietic stem cells which generate progenitor cells that are irreversibly committed to one of the various differentiation lineages. Recent studies in mice have shown that these hemopoietic stem cells can be isolated in homogeneous conditions. Purified human hemopoietic stem cell offer enormous therapeutic potential in allogeneic and autologous bone marrow transplantations. Furthermore the availability of such cells will enable crucial studies of normal and abnormal stem cell regulation as well as the development of gene therapy protocols. The long-term goal of this project is to define conditions that will sustain proliferation and self-renewal of highly purified human hemopoietic stem cells. The specific aims of this proposal are threefold. The first is to refine small scale stem cell purification procedures using flow cytometry and cell sorting. For this purpose it is proposed to prepare detailed maps of cell surface antigens expressed on cells that co-express CD34 using directly labelled anit-CD34 monoclonal antibodies and multiparameter flow cytometry and cell sorting. Selected cell fractions will be assayed in colony-forming assays and long- term bone marrow culture. Stem cells will be operationally defined as cells capable of initiating long-term bone marrow cultures as shown by their production of colony-forming cells after 5 weeks in culture. The second specific aim is to establish small and large scale stem cell purification procedures using immunoabsorption techniques. This is important because flow cytometry and cell sorting may not provide a practical approach for the preparation of purified stem cells for clinical purposes. These studies will further explore the use of bispecific anti- CD34 x anti-desferal tetrameric antibody complexes and columns containing desferal coated glass beads for the purification of cells that express CD34. Research in this area has the potential to yield relative simple procedures for both small and large scale (up to 1010 cells/column) procedures for the purification of (sub)populations of cells that express CD34 for laboratory and clinical studies. The last specific aim is to define culture conditions for the survival, activation and proliferation of sorted stem cell (sub)populations. For these studies low numbers or individual cells will be directly sorted into the wells of culture plates containing different feeder cells, growth factors, extracellular matrix proteins or antibodies. The fate of the cells during culture will be analyzed using a Cell Analyzer System that allows time lapse video recordings of microscopic images. Culture assays and monoclonal antibodies will be used to characterize the cells derived from the cultured sorted cells. Taken together these investigations in complementary areas of stem cell research will yield technology that is directly applicable in clinical medicine. The results will result in a better understanding of stem cell biology and new directions for further study.